The objective of this proposed research is to determine the origin of intramitochondrial pyridine nucleotides. This goal will be approached first by reinvestigating the uptake of NAD ion by intact liver mitochondria using 14C-NAD ion labeled in both the nicotinamide and adenine moieties in order to rule out the possibility that the previously observed uptake of NAD ion by mitochondria could be due to a cleavage of NAD ion to nicotinamide exterior to the matrix followed by uptake and resynthesis of nicotinamide into NAD ion. The second facet of this work will be investigation of the pathway for the biosynthesis of intramitochondrial NAD ion from nicotinamide. The fate of 14C-nicotinamide will be investigated in intact respiring mitochondria and in mitochondria depleted of ATP by preincubation with uncoupling agents. These experiments are designed to determine whether the first step in mitochondrial NAD synthesis from nicotinamide is conversion to nicotinic acid of NMN since the later reaction requires ATP while the former does not. Mitochondrial extracts will also be investigated for the presence of both the enzymes for the de novo synthesis of NAD via quinolinic acid and for the salvage pathway enzymes nicotinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase. These enzymes will then be purified and characterized sufficiently to determine if they differ markedly from the corresponding cytoplasmic enzymes.